Three-dimensional cell growth assay

ABSTRACT

The invention relates to devices and methods for growing cells in vitro in an enclosed device that allows for a three-dimensional measurement over time of both their proliferative and/or invasive properties. By growing the cells in an enclosed matrix that resembles the environment that the cells confront in vivo, the cells can divide, invade, and form branched networks as they do in living tissue, e.g., in an individual. The devices of the invention include a test chamber in which cells, e.g., tumor cells, are placed and permitted to divide and/or invade. Cells can be placed within an insert within a chamber of the device. A delivery chamber that connects to the test chamber enables the delivery of agents that can be studied, e.g., for their therapeutic potential. The assay devices of the invention can be used as model systems to study cancer biology and to evaluate the efficacy of anti-cancer therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/758,644, filed Jan. 11, 2001, issued as U.S. Pat. No. 6,602,701 onAug. 5, 2003, which claims priority from U.S. Provisional ApplicationNo. 60/175,616, filed Jan. 11, 2000. These prior applications areincorporated herein by reference in their entirety.

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

This invention was made with Government support under NIH/NCIR21CA84509-01 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to devices and methods for analyzing cellulargrowth in vitro.

BACKGROUND OF THE INVENTION

Tumors grow through two primary processes: proliferation and invasion.Proliferative growth represents the increase in size of the centraltumor mass through the division of cells. Invasive growth occurs intissues in the regions adjacent to and around the central tumor mass. Inthe invasion process, individual tumor cells detach from the centraltumor mass and begin to actively move through the surrounding,non-tumorous tissue, either by compression or enzymatic degradation. Thepresumably highly branched cells formed by these invading cellsrepresent a dynamically evolving network pattern. The chains formed inthe invasive network can be as thin as a single cell in width. Further,the invading cells are significantly elongated along the direction inwhich they are traveling.

Malignant tumors such as highly malignant brain tumors (e.g., gliomasand glioblastoma multiforme) have several features such asproliferation, invasion, central necrosis/apoptosis andneo-vascularization. Tumors outside the central nervous system showextensive metastasis by way of the blood circulatory and lymphaticsystems.

Several in vitro assays have been described that are designed to measureeither cell proliferation, migration, or invasion. For example, a cellcolony/spheroid-agarose assay uses cell suspensions within, ormulticellular tumor spheroids placed on top of agarose (in a cellculture dish), which is then covered with cell culture medium. CarlssonJ., Int. J. Cancer, 20:129-136, 1977. The assay is designed to study thegrowth dynamics of the cell colonies or spheroid. The medium superlayerdistributes growth factors and growth limiting factors produced by thetumor, without preserving their important regional concentrationdifferences (i.e., higher concentration around the tumor). Also, thesuperlayer has to be changed routinely, altering the environmentalsetting.

A 2D migration assay can be used to describe the movement of a cellpopulation from a central area in an expanding circle. Giese A.,Neurosurgery, 37:294-302, 1995. The cells are placed in a cell culturedish and covered with medium. Aside from the edge of the dish, there isno mechanical confinement and no chemo-gradients can be established dueto “equalizing” in the medium superlayer.

Invasiveness assays (e.g., commercially available through Costar® as the24-well Transwell™ System) use a medium-supplemented, two-chambersystem, which is designed to detect cell migration between the twochambers. Repesh L. A., Invasion Metastasis, 9:192-208, 1989. The insert(top chamber) has a polycarbonate membrane on the bottom, which has anumber of pores (8 μm size). After a certain number of cells are placedonto this membrane, they start to move through the pores and drop intothe lower chamber where they either start anchorage-independent growthor eventually attach. After an observation period the cell number inboth chambers is counted (Coulter Counter System®) and a ratio indicatesthe specific invasive potential of the cell line used. A variation ofthe assay uses a Matrigel® layer on top of the polycarbonate membrane toinvestigate the enzymatic activity of the cells to digest their waytowards the pores.

A spheroid-fetal rat brain aggregate assay uses rat brain aggregatesco-cultured on a medium/agar-layer and covered with a cell-culturemedium that is changed routinely. Khoshyomn S., J. Neuro-Oncology,38:1-10, 1998. The migration capacity of the tumor cells is determinedby the destruction of the rat brain aggregate, not by the directmeasurement of cell branches.

SUMMARY OF THE INVENTION

The invention is based on the discovery that cells can be grown in vitroin an enclosed device that allows for a three-dimensional measurement ofboth their proliferative and invasive properties. By growing the cellsin an enclosed matrix that resembles the environment the cells confrontin vivo, the cells can divide, invade, and form branched networks asthey are thought to do in living tissue, e.g., in an individual.Propagating the cells in vitro in this manner allows for the imaging andtemporal-spatial analysis of cells and cellular behavior that cannot beeasily achieved when the cells are grown inside an organism. The methodsand devices of the invention are particularly useful for studying thegrowth of tumor cells in vitro. The assay devices of the invention canthus be used as model systems to study cancer biology and to evaluatethe efficacy of anti-cancer therapeutics.

In general, the invention features an assay device for measuring theproliferation and/or invasion of cells, e.g., tumor cells. The deviceincludes a test chamber and a first delivery chamber arranged to contactthe test chamber. The device can also include a control chamber, e.g.,arranged to contact the first delivery chamber or a second deliverychamber. The first delivery chamber includes a wall with an opening toenable fluid communication between the first delivery chamber and thetest chamber. The device also includes a hollow cylinder enclosing alumen and arranged within the first delivery chamber, the cylinderincluding a wall with a hole that can be aligned with the opening in thefirst delivery chamber wall to enable fluid communication between thecylinder lumen and the test chamber.

The assay device can further include a cover that sealingly contacts thedelivery chamber, the test chamber, and the control chamber, if present.The assay device can also include a moveable interior wall that isarranged within the test chamber to be moved laterally within the testchamber, e.g., by turning screws located in holes in an outer wall ofthe test chamber. The assay device optionally includes a second moveableinterior wall that is arranged within the control chamber to be movedlaterally within the control chamber, e.g., by turning screws located inholes in an outer wall of the control chamber.

The invention also features an assay device that includes a plurality ofcylinders, each having a hole that can be aligned with the opening inthe delivery chamber wall to enable fluid communication between thecylinder and the test chamber, wherein the cylinders are interchangeableand each has a hole of a different size. In addition, the assay devicecan also include a second delivery chamber arranged to contact thecontrol chamber, e.g., in the same manner that the first deliverychamber is arranged to contact the test chamber. This would allow thecontrol chamber to be exposed to a control fluid, as compared to a testfluid in the test chamber. The control chamber can include a moveablewall that is arranged to move within the control chamber.

The test chamber of an assay device can include an outer wall with anopening to enable fluid communication between the test chamber and theexterior of the assay device. An assay device can also include a hollowinsert constructed to fit within the test chamber. The hollow insert cancontain a moveable wall that is arranged to move within the insert.

In another embodiment, the invention features an assay system formeasuring the proliferation and/or invasion of cells. The assay systemincludes an assay device of the invention, a pump having an input and anoutput, a first conduit that connects one end of the cylinder to thepump input, and a second conduit that connects a second end of thecylinder to the pump output to permit flow of fluid, e.g., a liquid orgas, from the pump, through the cylinder in the delivery chamber of thedevice, and back to the pump.

The assay system can also include an injection port connected to aconduit that permits the introduction of substances into the system,e.g., by microinjection. Additionally, the assay system can include adevice of the invention that includes a first moveable interior wallthat is arranged within the test chamber to be moved laterally withinthe test chamber, e.g., by turning screws located in holes in an outerwall of the test chamber. Furthermore, the assay system can include adevice of the invention including a second moveable interior wall thatis arranged within a control chamber to be moved laterally within thecontrol chamber, e.g., by turning screws located in holes in an outerwall of the control chamber. The advancement of the walls allows for acontinuous controlled increase of the mechanical confinement within thechambers and the study of its structural and functional impact on thedistinct features of the cell system (e.g., proliferation and/orinvasion).

The test chamber of an assay device of an assay system can include anouter wall with an opening to enable fluid communication between thetest chamber and the exterior of the assay device. An assay system canalso include a hollow insert constructed to fit within the test chamber.The hollow insert can contain a moveable wall that is arranged to movewithin the insert.

In another aspect, the invention features a method for detecting theproliferation of cells, e.g., tumor cells. This method includes thefollowing steps: (1) placing one or more cells in a matrix within thetest chamber of a device of the invention; (2) placing the device underconditions that permit the growth of the cells contained therein; (3)aligning the hole in the wall of the cylinder and the opening in thewall of the first delivery chamber to enable liquid medium to flow intothe test chamber; (4) flowing liquid medium through the cylinder withinthe delivery chamber of the device; and (5) evaluating the proliferationof the cells within both the test chamber. The test chamber can includea moveable wall and the method can include moving the moveable wall. Inone example, the placing of the one or more cells in a matrix within thetest chamber includes placing the one or more cells within a matrix in ahollow insert, and placing the hollow insert within the test chamber.The insert can include a moveable wall and the method can include movingthe moveable wall.

The method can also include flowing a gas through the cylinder withinthe delivery chamber of a device of the invention. The method canevaluate the proliferation of the tumor cells by counting the numbers ofcells within the control and test chambers, e.g., by microscopicanalysis of cells within the device. The tumor cells can optionally beextracted from the device for analysis.

The invention also features methods for detecting the proliferation oftumor cells wherein therapeutic agents are in the liquid medium thatflows into the test chamber of the device. Additionally, according tothe methods of the invention, therapeutic agents can be delivered to thetest chamber of the device through holes in an outer wall of the testchamber. The methods also include the step of flowing liquid medium fromthe test chamber through a hole in the outer wall of the test chamber tothe exterior of the device.

In another embodiment, the invention features a method for detectinginvasion of tumor cells. The method includes the following steps: (1)placing tumor cells in a matrix within both the test chamber of a deviceof the invention; (2) placing the device under conditions that permitthe invasion of the cells contained therein; (3) aligning the hole inthe wall of the cylinder and the opening in the wall of the deliverychamber to enable liquid medium to flow into the test chamber; (4)flowing liquid medium through the cylinder within the delivery chamberof the device; and (5) evaluating invasion of the cells within the testchamber. The method also includes flowing a gas through the cylinderwithin the delivery chamber of the device. The method can evaluate theinvasion of tumor cells by microscopic analysis of cells within thedevice. The tumor cells can be extracted from the device for analysis aspart of the method.

The invention also features methods for detecting invasion of tumorcells, wherein therapeutic agents are included in the liquid medium thatflows into the test chamber of the device. Additionally, according tothe methods of the invention, therapeutic agents can be delivered to thetest chamber of the device through holes in an outer wall of the testchamber.

The assay devices of the invention allow for dynamic three-dimensionalmeasurements of the proliferation and invasion of multicellular systemssuch as tumor cells in an in vitro assay. The lack of a mediumsuperlayer allows for some of the devices to be tilted to any angle andeven flipped 180 degrees for true three-dimensional measurement, withoutan alteration of the internal environment. The new assay devices of theinvention have the advantage of being able to investigate tumors asmulti-featured systems.

The invention also provides advantages over in vivo assays. Single cellinvasion cannot be easily studied in situ or in vivo (e.g., in animalmodels) because of the limitations of the resolution threshold ofimaging methods. The new assays allow for in vitro growth of cells,e.g., tumor cells, in three dimensions over time so as to mimic tumorgrowth in vivo, but also render the cells accessible to a wide range ofin vitro techniques that allow the study of their biology and theirreaction to various agents, including potential therapeutics. The newassay systems allow for a continuous flow through gel-embedded cells andthus allows for repeated measurements of cells as they grow in athree-dimensional environment. The devices described herein allow foreasier focusing on a tumor using microscopic techniques, thus increasingthe ability to achieve sharp pictures from all angles. The advanced, newin vitro models also limit the amount of necessary in vivoexperimentation by providing a pre-evaluation and a focus on promisingdrugs.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, suitable methods and materialsare described below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of a conflict in terminology, the presentspecification will control. In addition, the described materials andmethods are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation (top view) of a three-chamberedassay device of the invention.

FIG. 2 a is a cross-sectional view of the delivery chamber and thecylinder of a device of the invention, in which a substance is permittedto flow from the cylinder into the test chamber.

FIG. 2 b is a cross-sectional view of the delivery chamber and thecylinder of a device of the invention, in which the cylinder has beenrotated to prevent flow from inside the cylinder into the test chamber.

FIG. 3 a is a schematic representation (side view) of a three-chambereddevice of the invention with a cover in place.

FIG. 3 b is a schematic representation (side view) of an assay device ofthe invention, in which the cover is removed and the ridge forattachment of the cover is visible.

FIG. 4 is a schematic representation (top view) of a three-chamberedassay device of the invention, in which a moveable wall is arranged tobe advanced towards the delivery chamber.

FIG. 5 is a schematic representation (top view) of a three-chamberedassay device of the invention.

FIG. 6 a is a schematic representation of an insert.

FIG. 6 b is a schematic representation of an insert with two screwspositioned on one side of the insert.

FIG. 6 c is a schematic representation of the insert of FIG. 6 b rotated90 degrees.

FIG. 6 d is a schematic representation of an insert within an insertholder.

FIG. 7 is a schematic representation (side view) of a chamber of adevice of the invention containing an insert.

FIG. 8 is a schematic representation of a single pump assay system ofthe invention.

FIG. 9 is a schematic representation of an assay system of the inventionattached to a flexible table.

FIG. 10 is a schematic representation of a dual pump assay system of theinvention.

DETAILED DESCRIPTION

The invention relates to devices and methods for growing cells in vitroin an enclosed device that allows for a three-dimensional measurementover time of both their proliferative and invasive properties. Bygrowing the cells, e.g., tumor cells, in an enclosed matrix thatresembles the environment that the cells confront in vivo, the cells candivide, invade, and form branched networks as they are thought to inliving tissue, e.g., in an animal or a human. Propagating the cells invitro in this manner allows for an analysis of cells, e.g., tumor cells,as multi-featured systems and promotes the development of computationalmodels based on experimental data. Devices described herein can be usedfor short term or long term experiments.

A central feature of the devices of the invention is that they include atest chamber in which cells are placed and permitted to divide andinvade. The devices of the invention can also include a control chamber.A delivery chamber that connects to the test chamber allows for thedelivery of agents that can be studied for their therapeutic potential.The assay devices of the invention can thus be used as model systems tostudy multicellular biological systems. For example, they can be used tostudy cancer biology and to evaluate the efficacy of anti-cancertherapeutics.

Construction of the Assay Device

A top view of a device 10 of the invention is shown in FIG. 1. Thisdevice has a first side wall 16, e.g., of about 8 to 10 mm in length,and a second side wall 18, e.g., of about 12 to 30 mm in length. Thedevice in the examples below is rectangular, but can be square, round,elliptical, or other overall shape, given that other dimensions ofdevice components are adjusted to conform to the device shape. The wallsof device 10 can be made of plexiglass, glass, or others material ofsimilar rigidity and transparency. Device 10 is divided into threechambers, a test chamber 14, a control chamber 12, and a deliverychamber 26, that can be arranged centrally to separate the other twochambers. The test chamber 14 is treated experimentally in assaysperformed using the device and the control chamber 12 is left untreated.By having a system with two tumor-containing chambers, one can set upboth experimental and control assays at the same time using the samelots of tumor cells, gel, and media, and thus limit the effect ofslightly different growth factor compositions and facilitating theset-up for both experiments.

Delivery chamber 26 is shown in FIG. 1 to extend from one side of thedevice to the other, contacting both the top and the bottom of thedevice 10. In FIG. 1, delivery chamber 26 separates the other twochambers 12 and 14, but can be arranged on either side of test chamber14. The only requirement is that delivery chamber 26 contacts the testchamber 14, to enable flow of liquids inside the delivery chamber intothe test chamber. If delivery chamber 26 is located for example on sidewall 17 of device 10, then a wall would be required to separate testchamber 14 from control chamber 12. As shown in FIGS. 2 a and 2 b, ahollow cylinder 22 can be located within delivery chamber 26. Cylinder22 can be round and can be made of plastic. Delivery chamber 26 has ahole 26 a in the wall (or walls) located between it and the test chamber14. Similarly, cylinder 22 has a hole 22 a located in its wall thatcorresponds in location to hole 26 a in the delivery chamber 26. Theseholes are arranged to permit the flow (arrow 24 in FIGS. 1 and 2 a) of asubstance into test chamber 14. Only when both holes 22 a and 26 a arealigned will the substance flow into the test chamber 14.

The size of hole 26 a in delivery chamber 26 is larger than that of hole22 a in cylinder 22 and can be customized. Cylinder 22 can be removed,for example with a miniature screwdriver, to customize the size of itshole. Thus, any desired changes in hole size, and thus flow rate andvolume delivered into the test chamber 14, can be achieved by modifyingcylinder 22. Additionally, a collection of cylinders 22 can be providedthat each have holes of different sizes, thus permitting an immediateselection of the appropriate hole size for different assays without theneed for any mechanical modifications to the rest of the device. Thecylinder 22 can optionally be operated from the top of the device byremoving the top of the device and aligning hole 26 a and 22 a, from theoutside, without having to unhook the connectors and/or tubes.

FIGS. 2 a and 2 b illustrate the rotation of cylinder 22 to control flowof material into the test chamber 14. In FIG. 2 a, the lumen insidedelivery chamber 26 contains cylinder 22. This cylinder 22 can berotated inside delivery chamber 26, and can be entirely removed. Whenput in place, cylinder 22 can be connected to the outside conduits andpump mechanism. As illustrated in FIG. 2 a, when holes 26 a and 22 a arelined up with each other, fluid can flow into the test chamber 14.Because hole 26 a of delivery chamber 26 is larger than hole 22 a ofcylinder 22, liquids can be made to flow into test chamber 14 at variousangles (arrows 24, 30, and 34) based upon the degree of lineup betweenthe two holes. FIG. 2 b illustrates the further rotation (arrow 30) ofcylinder 22 such that hole 26 a and hole 22 a are no longer aligned.This prevents the flow of substances from inside cylinder 22 into testchamber 14. In addition to fluids, the assay system can also be arrangedto deliver a gas to test chamber 14 when the holes in delivery chamber26 and cylinder 22 are aligned. For example, various concentrations ofO₂ and CO₂ can be used to evaluate the dependence of partial pressure onthe cell system. Additionally, the assay system can be constructed todeliver a fluid and gas combination, e.g. to mimic the conditionspresent in normal blood flow.

In addition to the flow of substances into test chamber 14, the device10 can also be constructed to deliver substances (e.g., fluid or gas) tocontrol chamber 12. This can be accomplished in the same manner as fordelivery to test chamber 14, e.g., by providing a delivery chamber andoptionally a cylinder having holes that may be aligned to permit flowinto the control chamber 12. If the substances to be delivered tocontrol chamber 12 and test chamber 14 differ, then separate input andoutput conduits and pumping systems can be arranged for two separatedelivery chamber and cylinder combinations that each connect to only asingle chamber, thereby preventing the cross-flow of substances intendedto be delivered to a single chamber.

As illustrated in the side view of the device 10 in FIG. 3 a, a cover 40is placed on top of the device so as to fit tightly and seal the device.A sealant, e.g., silicon glue, can be used to attach the cover to thedevice. Preferably, the sealant can easily be removed and thus permitsthe reuse of the device. As shown in this side view, side walls 42 ofthe device can have a height of about 4 to 10 mm. Because the device canhave a tight fitting cover 40, and has no air holes, the possibility ofcontamination of the cells growing inside the device is relativelysmall. The addition of antibiotics to the gel/medium mixture alsofunctions to minimize the possibility of contamination. The mechanismfor attaching the cover 40 is further described in the side view of FIG.3 b. A ridge 50 extends above the top of three of the walls of thedevice. The top of ridge 50 has an overhang 52 that receives an edge ofthe cover 40 and holds it tightly in place. This ridge 50 functionsessentially like a frame. By extending above three of the four sidewalls of the device, ridge 50 permits cover 40 to slide in place toprovide a tight fit between cover 40 and device 10.

Moveable Interior Wall

As illustrated in FIG. 4, the new device can also include a moveableinterior wall 60. The moveable wall can be made of plexiglass, glass, orother such rigid material. The wall can be included in one or bothchambers 12 and 14. FIG. 4 illustrates the movement of wall 60 in testchamber 14. Wall 60 is advanced by two screws 62 in two holes 63 in theside wall 17 of the device. These screws can be made of, for example,plastic. The turning of the screws causes wall 60 to move towardsdelivery chamber 26 in the direction of arrow 61. Wall 60 is positionedwith respect to the interior surfaces of the device and the screws so asto remain parallel to delivery chamber 26 as it is advanced. Movement ofthis wall increases the mechanical confinement pressure inside the testchamber 14. When test chamber 14 contains a gel, this movement creates aregional pressure gradient, specifically an increase in pressure in thearea closest to moveable wall 60.

In the device illustrated in FIG. 4, wall 64, which is arranged not tomove, is located at one end of the device. Wall 64 can be fixed to theside 16 of the device by fitting screws that do not permit anadvancement of the wall to the interior of chamber 12. Alternatively,this chamber can completely lack a wall and holes 66 and instead besealed by side wall 16 of the chamber. In another alternative, holes 66can be fitted with screws similar to screws 62, which can be used toseal holes 66, or to move wall 64 towards delivery chamber 26.

Inserts for Assay Devices

A top view of a device 90 of the invention is shown in FIG. 5. Thisdevice has a first side wall 92 and a second side wall 94. The walls ofthe device can be made of the rigid, transparent materials describedherein, e.g., plexiglass. As in the device 10 depicted in FIG. 1, device90 is divided into a test chamber 14, a control chamber 12, and adelivery chamber 26. The delivery chamber 26 is designed in the samemanner as the delivery chamber of device of FIG. 1. Device 90 containsinserts 96 a and 96 b that can contain cells and a matrix. In FIG. 5,inserts 96 a and 96 b are placed in each of the test and the controlchambers. Each of the inserts is surrounded by a layer of media on allsides when the device is used. A device can optionally be constructedand used containing an insert in only one chamber. In one example, thewalls of the device are made of plexiglass and are 1 mm thick.

The use of an insert and insert holder, as described below, isparticularly advantageous when removal of the cell/matrix mixture fromthe device is desired. Insert 96 a functions as a removable containerthat holds cells and a matrix. In FIG. 5, insert 96 a is depicted as acube, though it can take any shape, e.g., rectangular, round, orelliptical. The insert contains at least one opening, e.g., a hole, topermit media to access the cells contained therein. For example, theinsert can contain holes on one side, e.g., the side that faces adelivery chamber.

The cube-shaped insert 96 a of FIG. 5 is depicted in more detail in FIG.6 a. The insert 96 a has holes 98 on all six sides, to allow media toaccess the interior of the insert 96 a from all sides. In some cases, aninsert may have an opening only on one surface, e.g., a surface facingthe flow from the delivery chamber. The insert 96 a has holes 98 thatpreferably are not large enough to permit escape of the matrix andcellular material from the insert. In one embodiment, the insert 96 a isa six-walled cube (4×4×4 mm) with plexiglass walls, e.g., 1 mm thick. Inthis embodiment, all six sides are perforated with holes (having adiameter of 0.1-0.3 mm), e.g., in a 1×1 mm grid. The insert can have aremovable top, e.g., a sliding or drop down top, that facilitates theplacement of cells into the insert and their removal therefrom. Theinsert is held in its x, y, z position in the chamber by bottom and topnodes, as described below.

Device 90 of FIG. 5 is designed so that the insert can be removed from achamber, e.g., a test chamber, by sliding back the lid of the device toanalyze cells contained within the insert. When removed from thechamber, the cells can be analyzed while in insert 96 a or followingtheir removal from the insert. When analyzing the cells while in theinsert, e.g., to perform online image analysis, the insert mayoptionally be placed in an insert holder. The insert holder functions tocontain the insert when removed from the device, while minimizing therate at which the gel dries, e.g., a surface of the insert holder coversone or more of the openings of the insert. The insert holder ispreferably manufactures with a transparent material, e.g., plexiglass orglass, as described herein. For example, when a cube-shaped insert isused, the insert holder can be four sided, with a removable top and/orbottom. The insert holder, with the insert contained therein, can thenbe subjected to various analyses, e.g., laser scanning confocalmicroscopy, or light microscopy.

In those devices that use an insert, a moveable wall can be placedwithin the insert to achieve the advantages described herein conferredby such a wall. For example, a moveable wall of an insert can beoperated by using screws that extend from outside the device, throughthe external wall, through the media in the chamber, and through a wallof the insert. As shown in FIG. 5, the screws 95, when turned, push onthe moveable wall and increase the pressure gradient within the interiorof insert 96 a.

If a moveable wall is not needed for a particular experiment, insert 96a can be sealed, e.g., using the short screws as described therein. Thedevice can be closed using a similar mechanism. If the moveable walls ofan insert have been used, e.g., advanced within the insert, then thescrews can be loosened to allow for removal of the insert from thechamber. The chamber walls can then be sealed, e.g., with 1 mm plugs.The screws can be placed back into the insert after its removal from thechamber.

FIGS. 6 b, 6 c, and 6 d depict an example of devices and methods for thehandling of an insert with a moveable wall, following the removal of theinsert from a chamber. In FIG. 6 b, screws 100 are repositioned in theinsert 96 a following the removal of insert 96 a from the chamber. InFIG. 6 c, the insert is turned 90 degrees with respect to FIG. 6 b. Ascale can be added to the insert, e.g., to the sliding lid of theinsert, e.g., by laser scratching. This scale can be used as a marker toensure that the moveable wall is at the same location when the screwsare repositioned as it was while the insert was in the chamber. If themoveable wall has changed position during the process of removing theinsert, then the wall can be returned to its proper position asindicated by the scale. Following the rotation of the insert in FIG. 6c, the insert can be placed in an insert holder 102, as depicted in FIG.6 d. The insert holder 102 of FIG. 6 d is a four sided device, with aremovable top 104 and a removable bottom. The removable top 104 is adrop down top that fits around the screws 100, so that the screws neednot be removed from the insert for the placement of the top. Followingan analysis of the material within the insert, the insert can be pushedthrough the sliding bottom of the insert holder and placed back in thetest chamber (by removing and repositioning the screws in the reverseorder of the steps described above).

A device can contain a node or nodes that prevent an insert from movingwithin a chamber. Nodes can be designed to permit media to access all ofthe sides of the insert. In device 114 depicted in FIG. 7, bottom nodes110 are affixed to the bottom of the chamber. Media 116 surrounds theinsert on all sides. Top nodes 112 are affixed to the top corner of theinsert 96. In this embodiment, the lid for the device is a slidable lidthat glides over the entire top of the device to make a seal. Asdepicted in FIG. 3 b, the lid slides over both chambers, andadditionally rests on the top nodes 112 of the insert 96. Nodes can beplaced on the lid, if the device is designed such that the nodes do notinterfere with the attachment of the lid to the device.

Chamber Outflow

A device of the invention can include a chamber outflow mechanism. Anoutflow can be incorporated into a test chamber, a control chamber, orboth. The device 90 of FIG. 5 contains a test chamber outflow 93. Thediameter of the outflow can be of the same size, smaller, or larger thanthe diameter of the removable connectors on the ends to the centralchamber. When used in combination with an insert in a device, theoutflow permits the continuous flow of media into a chamber, around thecells in the insert, and out of the chamber. The outflow can optionallybe connected to a continuous pump system, as described below.

Connecting the Device to a Pumping System

As illustrated in FIG. 8, devices 10 and 90 can be attached to a pump70, e.g., a continuous peristaltic pump, via conduits, e.g., tubing, 76.A switch 74 can also be used in the assay system 78. The peristalticpump 70 can be a low flow pump, having a flow rate of 0.03-8.2 ml/min(Fisher). Alternative pumps that can be used in the invention include anultra low flow pump (0.005-0.9 ml/min) and a medium flow pump (4.0-85.0ml/min). Peristaltic pump 70 creates a continuous flow that can bevaried as desired to permit the delivery of fluid and or gas to testchamber 14. As shown in FIG. 9, the assay system 78 can be placed on atable 80 that can be rotated 84 as desired, e.g., if flexible/rotatinglinks in the tubing prevents twists. Clamps 82 keep the conduits 76 inplace regardless of the angle of the table 80. Any leakage in the systemcan be measured, e.g., by the use of fluorescent spheres (for example,1-2 μm in size). The entire assay system 78, including the peristalticpump 70, can be placed inside an incubator and/or placed next to amicroscope. A direct AC-adapter can be used for the pumps of the assaysystem. Alternatively, a battery, e.g., a 9 or 12 volt battery, can beused in the pump 70.

In FIG. 10, a device 110 is attached to two peristaltic pump systems.Pump 112 circulates media through the delivery chamber 114 and into thetest chamber 116. Pump 118 effects the outflow of media (and optionallywaste products) from the test chamber 116. In this system, reservoirsare used for continuous supply (reservoir 120) and continuous removal(reservoir 122). The reservoirs can be equipped with filters, e.g., toavoid vacuum and contamination. The system can be constructed, and thepumps can be adjusted, so that the outflow velocity of the test chamberis higher, lower, or equal to the inflow velocity. Although FIG. 10depicts a non-continuous two pump system, a continuous system is alsoincluded within the invention.

Measurements of the fluid content of the outflow 124 can provideinformation as to metabolism within the cell system in test chamber 116.In addition, the flow in the device 110 can be reversed to expose cells,e.g., cells in the test chamber 116, to conditioned medium. The flowfrom pump 112 and/or pump 118 can be reversed. Measurements ofmetabolites can also be conducted in the control chamber 126, e.g., inthe same manner as for the test chamber 116 or by microsampling throughthe threads which hold the wall-operating screws.

Because the chamber can be constructed to lack air holes, it may not benecessary to place the system 78 inside of a CO₂ incubator. Rather itmay be necessary to only ensure that device 10 remains heated as isrequired for maintenance of the cells therein. Therefore, system 78 canbe potentially operated outside of an incubator in an environment thatwarms device 10. For example, the device 10 can be placed inside of aheated plexiglass chamber that contains either the entire system 78 orthat contains only the device 10 and has holes in the sides of theplexiglass that allow the conduits to enter and exit the chamber andconnect with the pump. The main requirement is that the medium or otherliquid or gas that is pumped by the pump 70 reaches appropriateculturing temperatures by the time it enters delivery chamber 26.

Nonetheless, for long-term experiments the entire device can be placedin an AC-equipped incubator, thus operating both pump-systems frominside. The battery mechanism allows the continuation of the flow whiletaking images.

A device described herein can be constructed to measure and/or adjustgas, e.g., O₂ and/or CO₂, concentrations within the device. For example,the system depicted in FIG. 10 can include a measurement device 113 afor pump system 112 and a measurement device 113 b for pump system 118.A measurement device can be included in one or both of the pump systems.In one embodiment, the measurement device detects the CO₂ concentrationand communicates with another device to ensure that the concentration ismaintained within an appropriate range, e.g. about 5% CO₂. The use ofsuch measurement and/or adjustment devices allows for the constructionand operation of an autonomous device, e.g., one that does not require aspecific atmosphere for the operation of the device. Devices containingmeasurement and/or adjustment devices are particularly useful for longterm cultures, e.g., those that use the inserts described herein.

In the system 110 depicted in FIG. 10, the measurement device 113 a islocated between the reservoir 120 and the inflow 117, but after theswitch 115 a. Likewise, the measurement device 113 b is located betweenthe reservoir 120 and the outflow 124, but before the switch 115 b,e.g., to allow measurements to be taken before samples are withdrawnfrom the system.

The substances that can be pumped through the assay system 78 include,for example, liquids (solutions or dispersions) such as medium (enrichedor deprived), growth factors, growth inhibitors, chemotherapeuticsubstances, radiosensitizers, and prodrugs as required for various genetherapy experiments, as well as gases, such as oxygen, carbon dioxide,and mixtures of gases. The substances can be added to the system via theinjection port 72. In addition to the injection port 72, plugs or screws62 (FIG. 4) can also be removed from holes 63 to supply substancesdirectly to the matrix inside test chamber 14 or into the insert 96 orthe medium around the insert. Specifically, Hamilton micro-syringes aswell as micropipettes can be used to deliver agents into the gel throughholes 63. These agents can include viral vectors, material formicroinjection, dye to study cell communication, or substances togenerate competing growth factor loci. Delivery of agents via the holes63 differs from delivery via holes 22 a and 26 a in the delivery chamber26 in that it permits delivery to a different site in the tumor as wellas the delivery of solid components. The holes 22 a and 26 a in thedelivery chamber are particularly well-suited to the delivery ofliquids, whereas the holes 63 permit the introduction of solidsubstances, for example by mixing the solid in a gel and injecting itthrough hole 63. Furthermore, delivery of a substance via a hole 63permits the use of fine techniques such as the microinjection of singlecells within the test chamber 14.

Culturing Cells in the Assay System

Both primary cells and established cell lines can be assayed using thissystem. Both tumor and non-tumor cells can be used. For example, tumorcells, endothelial cells, stem cells, and a variety of tissue specimensand co-cultures can be propagated in the devices of the invention. Thecells to be studied can optionally be cultured in vitro prior to beingplaced in the assay device. Standard media and incubation conditions canbe used.

It is expected that a variety of tumor cells can be tested in the assay.These include various brain tumors, breast tumors and lung tumors.Preferably, tumor cell spheroids are collected to be used in the device.Many glioma and non-glioma cell lines form spheroids, and both spinnerflask methods as well as agarose medium superlayer methods enable theformation of spheroids from cell suspensions.

Spheroids can be washed to remove serum and then placed in a gel, suchas Matrigel® (BIOCAT®, Becton Dickinson, Franklin Lakes, N.J.). Althoughthe composition of the gel is important (e.g., the gel consistencypermits tumor cells to proliferate and invade the gel matrix), it neednot necessarily be growth factor-reduced Matrigel®. The gel can be mixedwith medium at a ratio of 3:1 (gel:medium). The medium to be used canvary widely. For example OPTI-MEM® or DMEM can be used with variousconcentrations of growth factors. Survival time of the spheroids can beextended by adding higher growth factor concentrations or by adding full(serum supplemented) DMEM. Similarly, if using an insert, the mediumcomposition in the circulation can be altered to influence the viabilityof the cells within the insert.

If not using an insert, and its associated exposure to a continuousreplenishment of nutrients, then experiments are typically stopped atabout 144 hours after inserting the multicellular tumor spheroid intothe device to diminish the effects of central multicellular tumorspheroid-quiescence and necrosis. This marks the time-point at whichvolumetric growth increase typically becomes insignificant. However,successful experiments have been performed in which cells were keptalive for more than 168 hours by using “full” or complete medium for thegel composition. The experimental setting can therefore be tailored to aspecific cell line, a specific commercially available or custom-madegel, or a specific time frame. Furthermore, variations in either gelcomposition or gel consistency enable the investigation of thedependence of growth patterns on environmental conditions. By using aninsert, the time frame will be significantly extended as a continuoussupply of nutrients through the pump systems allows for longer in vitrocell growth periods.

The effect of various substances on cell growth can be evaluated byproviding the substance either in the original gel matrix, adding thesubstance via one of the holes 63 of the device into the gel or, if aninsert is used then also into the medium of the chamber, or by pumpingthe substance through the cylinder in the delivery chamber and allowingit to enter the test chamber 14 by way of holes 22 a and 26 a. Anysubstance that can enter the system through one of these mechanisms canbe tested with respect to its impact on the system. These substancesinclude, for example, growth factors and anti-cancer therapeutics. Theoutflow reservoir 122 can be used to determine the metabolic impact ofthese substances on the cell system.

Radiation studies, such as Boron Neutron Capture Therapy (BCNT), canalso be performed on cells, e.g., tumor cells, in the assay system. BCNTentails the irradiation of boron-10 (¹⁰B), a non-radioactive stableisotope with low-energy thermal neutrons, in order to yield high linearenergy transfer particles ⁴.He and recoiling ⁷Li. Since it is preferredfor BCNT to localize a high number of ¹⁰B atoms on or within theneoplastic cells, growth factors can be tagged with ¹⁰B. For example, alimited amount of growth factors (e.g., 50-100 ng/ml EGF/TGF-α) can beimplanted or injected to effect a regional impact on the invasivesystem. Growth factor-loaded agarose-gel particles can be used in theassay. Similarly, ¹⁰B can be injected locally within the assay toevaluate its selective cell killing ability. Furthermore, growth factorsand ¹⁰B can be co-implanted or co-injected at the same site in acombined approach. The growth factors and ¹⁰B can also be incorporatedin polymer platelets by applying technology currently used fordrug-wafers.

Analysis of Cell Growth in the Assay Device

The new assays are designed to permit the study of cells, e.g., tumorsas complex, dynamic, self-organizing biosystems, i.e., the study of theadaptive interdependency of these key features. The interdisciplinaryapproach includes tumor biology, bioengineering, mathematical biology,physics, materials science, computational science, and complex systemsscience. The approach is directed to ultimately develop an array ofcomputational models based on experimental data. To gain those datasets, one needs to develop novel experimental settings, preferentiallyonly one, that are capable of describing tumors as multi-featuredsystems.

After the culture period in the device has been completed, the entiregel cube can be fixed and preserved for histological analysis.Specifically, formalin is added to the chamber, the whole gel is fixed,and a cube with the cell, e.g., a tumor cell, in place or an insert isextracted from the chamber. This entire cube is embedded in paraffin andcan then be divided into microsections. Classical pathologicalevaluations can be performed on the sections, includingimmunohistochemistry, DNA analysis (both genetic and epigenetic), andadvanced Laser Capture Micro-Dissection (LCM), all using standardtechniques, to search for regional genetic and epigenetic changes.

Proliferation of cells in the device can be measured by varioustechniques. For example, cells taken from the device can be digestedwith trypsin to make a single cell suspension and then counted (e.g.,using a Coulter Counter) to determine the extent of proliferation thathas occurred since the start of the assay. Additionally, a morphometricmeasurement can be taken that measures the volume of a cell mass as ameans to calculate the total cell number.

Invasiveness of cells in the device can be measured by varioustechniques, such as statistical and fractal analysis, mathematical andcomputational methods combined with advanced imaging analysis, as wellas histological methods.

Cells can also be extracted from the gel for further growth eitherduring the culturing period or at the end of the culturing in the assaydevice. The reculturing of cells that have been extracted from thedevice allows for their expansion to further characterize various celltypes of interest. Cells can be re-cultured using a specificgel-digesting enzyme such as Dispase or MatriSperse (BIOCAT/BectonDickinson). This enables the analysis and or expansion ofsub-populations from different regions of the biosystem.

Cells within the device can also be examined as the assay is in progressfor 3D analysis over time, without unhooking the device from thecontinuous pump. For example, light microcopy can be used to takephotographs of tumor cells. The size and mobility of the table uponwhich the device rests allows it to fit under most microscopes.Photographs can be taken at time intervals and can be taken at specificpositions in the device by returning the microscope to a designatedlocation. Video time-lapse microscopy can also be performed, optionallyusing a laser to scratch an x, y, z grid on a surface of the device thatallows the use of coordinates on the device itself (rather thancoordinates on a microscope) to find and examine specific locations, andtherefore obviates the need to use the same microscope for allmeasurements.

Fluorescent techniques can also be used to evaluate cell growth. Thecells can be pre-stained with a fluorescent substance before beingplaced inside the gel. Confocal laser scanning microscopy can then beused to analyze the cells in a three-dimensional setting. Some of thefluorescence intensity can be lost in this system as a result ofdilution of the fluorescent substance by cell division and by bleaching.However, in some circumstances the fluorescent substance can bereplenished by its addition to the assay system while the assay is inprogress using the routes described earlier. Alternatively, the cellscan be genetically engineered before or during the assay to express agene encoding a fluorescent product, such as green fluorescent protein(GFP) or red fluorescent protein (RFP). The gene encoding a fluorescentproduct can be delivered to a cell for example by a retroviral vector,which can be added later when an experiment is in progress, e.g., usingthe routes described earlier.

Advanced imaging techniques can also be used to analyze cells with inthe device. The assay fits the needs for microstructural analysis of thegel with microtomography, microMRI, and/or a Synchrotron. Thesetechniques permit the analysis of any voids and solid structures withinthe gel. This analysis is important to study the mechanisms of cellinvasion such as the principle of “least resistance, most permission andhighest attraction.”

The insert holder allows these image-analyses of the cell-gelcomposition even when the insert is used. In that case, the placement ofthe insert into the insert holder can reduce the thickness of thematerial which has to be imaged as compared to image the insert withinthe chamber.

EXAMPLES

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1 Culture of the Human U87MGmEGFR Glioblastoma Multiforme CellLine as a Spheroid

The human U87MGmEGFR glioblastoma multiforme cell line was cultured inDMEM (GIBCO BRL, Life Technologies™, Grand Island, N.Y.) supplementedwith 10% heat inactivated cosmic bovine serum (HyClone®, Logan, Utah)and 400 μg/ml in G418 (Life Technologies™) in a humidified atmosphere of5% CO₂ at 37° C. Compared to the wild-type epidermal growth factorreceptor (wtEGF-R), these stable-transfected cells co-express also anEGFR variant (mEGFR=ΔEGFR, EGFR_(vIII), (2×10⁶/cell)). This specificmEGFR has an in-frame deletion of 801 bp of the coding sequence for theexternal ligand-binding domain, rendering the receptor constitutivelyactive and incapable of signal-attenuation by down-regulation. Thisgenetic rearrangement is rather common in glioblastoma multiforme tumorsand in vitro the mutation confers enhanced tumorigenicity by increasingproliferation and reducing apoptosis. Due to a yet unknown mechanismU87MGmEGFR cells rapidly form multicellular tumor spheroids (MTS) inculture after reaching monolayer confluency and detach at a certain sizedue to the laminar flow dynamics of the surrounding medium.

The floating U87MGmEGFR MTS were collected with Pasteur-pipettes andwashed gently in OPTI-MEM® (GIBCO BRL) to eliminate residual serum.Using Pasteur-pipettes the spheroids (≈500-700 μm in diameter,(0.7-1.0×10⁴ U87MGmEGFR cells)) were then placed in between two layersof growth factor-reduced matrix, Matrigel® (GFR-M) (BIOCAT®, BectonDickinson, Franklin Lakes, N.J.), which forms a reconstituted basementmembrane at room temperature. Initially extracted from theEngelbreth-Holm-Swarm mouse tumor, this specific matrix variant containsless than 0.5 ng/ml EGF and 1.7 ng/ml TGF-β (as well as 61% laminin and30% collagen IV) as compared to the commonly used full-Matrigel. We thenreduced these growth factors and extracellular matrix (ECM) proteinseven further by supplementing the gel with reduced serum mediumOPTI-MEM® at a ratio of 3:1 GFR-M to medium. This assay was performed ina completely enclosed 1 cm³ plexiglass cube, having no input/outputconnections or openings to a delivery chamber. The total GFR-M/OPTI-MEM®volume per well of the 1 cm³ device was about 2 ml per chamber. Todiminish the effects of central multicellular tumor spheroid quiescenceand necrosis, the experiments were stopped after 144 hours postmulticellular tumor spheroid placement. This marks the time-point whenthe volumetric growth increase becomes insignificant. The one-day changein volume drops below 15%, which equals less than half the valuesobtained during the steep growth phase (≈30% between 48 and 96 hours),and therefore signals the onset of the decelerating growth phase.

Example 2 Invasive Network Developing in the Three-dimensional Assay

The human U87MGmEGFR glioblastoma multiforme cell line was cultured in a1 cm³ plexiglass cube device as described in Example 1, except that DMEMfull medium (serum supplemented) was used. This enriched medium allowsfor an extended growth of the cells in the assay. The multicellulartumor spheroid (U87MGmEGFR) was analyzed at time 24 hours and at time120 hours. The invasive cells were seen to extend the initiallyestablished branches. A darkened structure, an attractor-tube, wasdetected which consists of a nutritive gel with a high consistency. Thelack of invasive branches toward this side along with the sustainedvolumetric expansion of the attached multicellular tumor spheroiddemonstrated the impact of this solid attractor on the tumor system. Byavoiding a higher consistency structure, the expanding invasive branchesappear to follow the proposed principle of “least resistance and mostpermission.”

Other Embodiments

It is to be understood that, while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications of the inventionare within the scope of the claims set forth below.

1. A method for detecting cell proliferation, the method comprising:providing a device comprising (a) a test chamber, (b) a first deliverychamber arranged to contact the test chamber, the first delivery chambercomprising a wall with an opening to enable fluid communication betweenthe first delivery chamber and the test chamber, and (c) a hollowcylinder enclosing a lumen and arranged within the first deliverychamber, the cylinder comprising a wall with a hole that can be alignedwith the opening in the first delivery chamber wall to enable fluidcommunication between the cylinder lumen and the test chamber; placingone or more cells in a matrix within the test chamber of the device;exposing the device to conditions that permit the growth of the cellscontained therein; aligning the hole in the wall of the hollow cylinderand the opening in the wall of the first delivery chamber to enableliquid medium to flow into the test chamber; flowing liquid mediumthrough the hollow cylinder within the delivery chamber of the device;and evaluating the proliferation of the cells within the test chamber.2. The method of claim 1, wherein placing the one or more cells in amatrix within the test chamber comprises placing the one or more cellswithin a matrix in a hollow insert, and placing the hollow insert withinthe test chamber.
 3. The method of claim 2, wherein the insert comprisesa moveable wall, and further comprising moving the moveable wall.
 4. Themethod of claim 1, wherein the test chamber comprises a moveable wall,and further comprising moving the moveable wall.
 5. The method of claim1, further comprising flowing a gas through the hollow cylinder withinthe delivery chamber of the device.
 6. The method of claim 1, whereinproliferation is evaluated by counting the numbers of cells within thetest chamber.
 7. The method of claim 1, wherein proliferation isevaluated by microscopic analysis of cells within the device.
 8. Themethod of claim 1, further comprising extracting tumor cells from thedevice and analyzing the extracted tumor cells.
 9. The method of claim1, wherein the liquid medium comprises a therapeutic agent.
 10. Themethod of claim 1, further comprising flowing liquid medium from thetest chamber through a hole in the outer wall of the test chamber to theexterior of the device.
 11. A method for detecting invasion of tumorcells, the method comprising: providing a device comprising (a) a testchamber, (b) a first delivery chamber arranged to contact the testchamber, the first delivery chamber comprising a wall with an opening toenable fluid communication between the first delivery chamber and thetest chamber, and (c) a hollow cylinder enclosing a lumen and arrangedwithin the first delivery chamber, the cylinder comprising a wall with ahole that can be aligned with the opening in the first delivery chamberwall to enable fluid communication between the cylinder lumen and thetest chamber; placing tumor cells in a matrix within the test chamber ofthe device; placing the device under conditions that permit the invasionof the cells contained therein; aligning the hole in the wall of thehollow cylinder and the opening in the wall of the first deliverychamber to enable liquid medium to flow into the test chamber; flowingliquid medium through the hollow cylinder within the delivery chamber ofthe device; and evaluating invasion of the cells within the matrix inthe test chamber.
 12. The method of claim 11, further comprising flowinga gas through the cylinder within the delivery chamber of the device.13. The method of claim 11, wherein invasion is evaluated by microscopicanalysis of cells within the device.
 14. The method of claim 11, furthercomprising extracting tumor cells from the device and analyzing theextracted tumor cells.
 15. The method of claim 11, wherein the liquidmedium comprises a therapeutic agent.
 16. The method of claim 11,wherein placing the tumor cells in a matrix within the test chambercomprises placing the tumor cells within a matrix in a hollow insert,and placing the hollow insert within the test chamber.
 17. The method ofclaim 16, wherein the insert comprises a moveable wall, and furthercomprising moving the moveable wall.
 18. The method of claim 11, whereinthe test chamber comprises a moveable wall, and further comprisingmoving the moveable wall.
 19. The method of claim 11, further comprisingflowing liquid medium from the test chamber through a hole in the outerwall of the test chamber to the exterior of the device.
 20. The methodof claim 11, wherein invasion is evaluated by counting the numbers ofcells within the test chamber.